Diagnosis of viral infection is carried out mainly by a method of detecting a virus or a virus-related component (protein or nucleic acid) or by a method of detecting a specific antibody produced by the living body upon viral infection.
Usually, infection with HBV can be known by confirming the presence of HBs antigen or HBc antibody. As an indicator not only for infection with HBV but also for a clinical state of an HBV carrier or judgment of prognosis and treatment efficacy, the amount of hepatitis B virus e antigen (HBe antigen), an antibody to the antigen, or DNA of HBV (HBV-DNA) is measured.
Among antigens constituting HBV viral particles (HBV particles), HBs antigen is a major constitutional envelope protein on the surface of infectious HBV particle and is anchored in a hepatocyte-derived lipid bilayer in which a core particle containing HBV-DNA is enveloped. In blood from a patient infected with HBV, there are noninfectious small spherical particles or tubular particles consisting of HBs antigens. The small spherical particles are present most abundantly in blood, and about 1000 small spherical particles are observed per one or several HBV particles. A majority of HBs antigen test agents commercially available at present mainly detects HBs antigen in the form of small spherical particles.
HBs antigen is a membrane protein consisting of 226 amino acid residues (amino acid numbers 1 to 226) and penetrating 4 times through a lipid bilayer. Although a model of the transmembrane structure of HBs antigen is not fully elucidated, Howard et al. (Howard et al., Viral Hepatitis and Liver Disease (ed by Zuckerman A J, Alan R), pp. 1094-1101, Liss Inc., New York, 1988) have proposed that the HBs antigen is composed of a (ER lumen side) region, outside a lipid bilayer, consisting of positions 1 to 11 from the N-terminal of HBs antigen, a hydrophobic transmembrane region penetrating through a lipid bilayer consisting of positions 12 to 28, a region inside the lipid bilayer consisting of positions 29 to 80, a hydrophobic transmembrane region consisting of positions 81 to 97, a hydrophilic ER lumen region consisting of positions 98 to 156, and two hydrophobic transmembrane regions consisting of positions 157 to 226 (FIG. 1).
A main common “a” determinant used in detection of HBs antigen in conventional methods is positioned in the amino acid positions 110 to 156, contained in amino acids in the positions 98 to 156 localized at the ER lumen side, that is, on the surface of viral particle. This common “a” determinant is reported to consist of a complicated higher-order structure wherein at least 4 epitopes are present (Hiroaki Okamoto, “Nippon Rinsho, Bunshi Kan-En Uirusubyogaku, Kiso-Rinsho-Yobo” (Japanese Clinic, Molecular Hepatitis Virology, Fundamental-Clinic-Prophylaxis), Lower Volume, Hepatitis A, B, D, E Viruses, pp. 212-222, published on Oct. 26, 1995).
HBV is DNA virus, but HBV is known to undergo mutation comparable with RNA virus because during viral proliferation, its DNA is replicated into RNA, and from this RNA, DNA is synthesized by reverse transcriptase. Accordingly, it is estimated that mutants having various kinds of mutations occur in an individual infected with HBV. When external selective stress such as neutralizing antibody is applied to the HBV in such individual, there occurs the phenomenon in which HBV strains sensitive to the stress are decreased, while mutants resistant or insensitive to the stress are increased. The so-called “escape mutant” coming to be problematic in recent years is a mutant which has undergone substitution, deletion or insertion of amino acid(s) in the main common “a” determinant, thereby being endowed with an ability to maintain its infection by escaping from an antibody recognizing the “a” determinant before mutation.
One problem associated with occurrence of the escape mutant is that this mutant can keep persistent infection because it can escape from an antibody induced by inoculation with a vaccine utilizing the “a” determinant before mutation.
Another problem is that this escape mutant cannot be detected in conventional HBs antigen examination methods. Generally, the escape mutant having a mutation on the common “a” determinant has lower reactivity with a monoclonal antibody against the common “a” determinant in the wild-type HBV, and thus a monoclonal antibody against the wild-type common “a” determinant, used in conventional HBs antigen examination methods, cannot recognize the HBs antigen in the escape mutant type HBV, thus failing to find actually occurring HBV infection. For example, it is reported that a 145Arg mutant, that is, the mutant wherein amino acid at position 145 was changed from Gly in the wild type to Arg, has significantly lower reactivity with a monoclonal antibody against the common “a” determinant (Hiroaki Okamoto, “Nippon Rinsho, Bunshi Kan-En Uirusubyogaku, Kiso-Rinsho-Yobo” (Japanese Clinic, Molecular Hepatitis Virology, Fundamental-Clinic-Prophylaxis), Lower Volume, Hepatitis A, B, D, E Viruses, pp. 212-222, published on Oct. 26, 1995). Actually, it is reported that transfusion of blood, shown to be HBs antigen-negative in a screening test of HBV by using the conventional HBs antigen measurement reagent, caused infection with HBV (Thiers et al. Lancet, ii, 1273-1276, 1988).
In acute infection with HBV, there is a reported phenomenon in which an HBV-infected patient is HBs antigen-positive in an initial stage of infection and then turns HBs antigen-negative and simultaneously becomes HBs antibody-positive. The reason that the patient becomes HBs antibody-positive is that an antibody against the common “a” determinant of HBs antigen is produced in the body of the patient. The patient's antibody against the common “a” determinant binds to the same region as in the common “a” determinant recognized by a monoclonal antibody used in the HBs antigen test reagent, thus leading to competition between both the antibodies, and by the competition, the sensitivity of the HBs antigen test reagent is reduced so that the detection of HBV by the test reagent is prevented.